Journal of Health Science Vol. 53(2007) No. 6

MINIREVIEW

Polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (NPAHs) are widespread carcinogenic compounds that arise from occupational, environmental and dietary sources. The metabolites of PAHs and NPAHs in biological fluids have been investigated as potential biomarkers for assessing human exposure to them, and, particularly, urinary metabolites are the excellent candidates due to the non-invasiveness and convenience of collecting the sample. Here we describe HPLC methods for accurately determining one type of these metabolites, monohydroxy PAHs (OHPAHs). The developed method was applied to the urine samples of non-smoker taxi drivers, traffic police officers and rural villagers of Chiang Mai, Thailand. The results showed higher urinary concentrations of OHPAHs in rural villagers, suggesting the higher respiratory exposure to PAHs contained in smoke from biomass burning. On the other hand, 1-nitropyrene (1-NP) is one of the most abundant NPAHs in diesel exhaust particulate matter (DEP). We also developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining urinary 1-NP metabolites. 1-NP metabolites were quantified in urine from healthy subjects. 6- and 8-Hydroxy-N-acetyl-1-aminopyrenes (OHNAAPs) and 6- and 8-hydroxy-1-nitropyrenes (OHNPs) were the most abundant 1-NP metabolites in human urine. The presence of OHNAAPs and OHNPs in human urine was demonstrated for the first time.

The neurotoxicity of Parkinson's disease (PD)-related tetrahydroisoquinolines (TIQs) and organotins is reviewed. PD is one of the most common neurodegenerative diseases among aged people and characteristized by the selective death of nigrostriatal dopaminergic neurons, but its pathological mechanism remains unknown. 1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ), an endogenous brain amine, was proposed as a possible PD-inducing neurotoxin, and its characteristics relevant to PD were evaluated. 1BnTIQ was detected in the mouse brain and human cerebrospinal fluid (CSF), and 1BnTIQ content was found to increase in the patients with PD. Repeated administration of 1BnTIQ induced PD-like symptoms in monkey and mice. 1BnTIQ induced dopaminergic cell death and subsequent dopamine decreases in organotypic slice co-culture, an in vitro culture system similar to the in vivo physiological environment. 1BnTIQ treatment also inhibited NADH-ubiquinone oxidoreductase in the mitochondrial respiratory chain. Thus, 1BnTIQ might a causative factor for PD, although the concentration required for neurotoxicity is higher than that found in the parkinsonian cerebrospinal fluid. We propose that tributyltin is another neurotoxin that acts through the glutamatergic pathway. Glutamate is one of the most abundant neurotransmitters in the central nervous system (CNS), but excessive release of glutamate causes prolonged stimulation of NMDA receptors, inducing calcium overload and neuronal death that is collectively referred to as excitotoxicity. Glutamate-mediated toxicity is selective for the CNS because only the CNS possesses this system. We clarified that tributyltin induces extracellular glutamate release and that tributyltin-induced neuronal death is mediated by glutamate excitotoxicity in cultured rat cortical neurons.

Tumour markers correlate strongly with prognosis on tumour burden. Glycoprotein components and lysosomal hydrolases play an important role in carcinogenesis. Hence, this study was launched to evaluate the effect of Kalpaamruthaa (KA), a modified Siddha preparation, on the changes in glycoprotein components, lysosomal enzymes and marker enzymes in control and mammary carcinoma bearing rats. A significant increase in the activities of alkaline phosphatase (ALP), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH) and 5' nucleotidase (5'-NT) in plasma, liver and kidney were observed in animals with mammary carcinoma. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly reduced in the liver and kidney whereas increased in plasma of cancerous animals. On administration of KA, these changes were reverted back to near normal levels. The increased levels of glycoprotein components (hexose, hexosamine and sialic acid) and in the activities of lysosomal enzymes such as acid phosphatase (ACP), β-D-Glucuronidase, β-D-Galactosidase, N-acetyl-β-D-glucosaminidase and Cathepsin-D (CD) found in mammary carcinoma were also significantly decreased in KA treated animals. In all these studies, simultaneous KA administration proved more efficacious than post KA treatment, thus depicting the effective control of KA against the development of mammary carcinoma.

In this study, we describe and adapt the relevant methods of computed tomography (CT) and stereology to estimate renal cell carcinoma (RCC) volume and volume ratio and compare the RCC volume estimations with the tumor stage. The study included 126 (82 men, 44 women) patients with RCC. The patients were evaluated by CT. The volume and volume ratio of the entire RCC was estimated by the following formula of Cavalierie's principles. According to TNM (tumor, nodes, metastasis) classification, there were 56 (44.4%), 30 (23.8%), and 40 (31.7%) cases in the stage T1, T2, T3, respectively. The results of the volume measurements which obtained from the Cavalier method were assessed according to the stages and were found as 125.52±102.18 (25-394) cm3, as 346.25±112.55 (181-545) cm3 and 694.88±405.46 (142-1546) cm3 in stage T1, T2 and T3, respectively. The volume ratios between the stages were compared statistically and a significant difference were found between the stage T1 and stage T2, stage T2 and the stage T3 and stage T1 and stage T3, respectively. The average tumor volume ratios was found as 28.44%±14.37% (8.69%-61.26%), 55.42%±12.73% (25.78%-73.86%), and 72.48%±17.15% (48.80%-97.15%) in stage T1, T2 and T3, respectively. The present evaluation of RCC volume can be done on any complete set of CT images, where plane scan distance and magnification factor is known, which already take place on to CT images.

The present study was designed to examine the cardioprotective effect of betaine on mitochondrial function in isoprenaline-induced myocardial infarction in rats with respect to changes in the mitochondrial energy status and antioxidant defense system. Prior oral treatment with betaine significantly prevented the isoprenaline-induced elevation in the levels of diagnostic maker enzymes [alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK)] and homocysteine in plasma of the experimental group of rats. Its administration significantly counteracted the isoprenaline-induced aberrations in the myocardial energy status by maintaining the levels of myocardial ATP and betaine contents and the activities of mitochondrial TCA cycle enzymes [isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KDH), succinate dehydrogenase (SDH), and malate dehydrogenase (MDH)] and respiratory marker enzymes (NADH dehydrogenase and cytochrome-c-oxidase) at near normalcy. It also exerted an antioxidant effect against isoprenaline-induced myocardial infarction by blocking the induction of mitochondrial lipid peroxidation (LPO). A tendency to minimize the isoprenaline-induced alterations in the level of reduced glutathione (GSH) and in the activities of glutathione-dependent antioxidant enzymes [glutathione peroxidase (GPx) and glutathione-S-transferase (GST)] and antiperoxidative enzymes [superoxide dismutase (SOD) and catalase (CAT)] in the heart mitochondria was also observed. The results of the present study indicate that the overall cardioprotective effect of betaine is probably related to its ability to maintain the myocardial energy status (ATP) at higher level by maintaining the activities of TCA cycle enzymes and respiratory marker enzymes at near normalcy, and/or to its free radical-scavenging ability against isoprenaline-induced lipid peroxidation, which is primarily responsible for the irreversible necrosis of the myocardial membrane.

Autoimmune hepatitis (AIH) serum contains autoantibodies against soluble liver antigen/liver and pancreas (SLA/LP), a protein that interacts with the selenocysteine (Sec)-tRNASec. We investigated the influence of AIH serum on Sec production. Two (#3 and #4) of four AIH sera inhibited its synthesis, with serum #4 showing the strongest inhibition. This did not parallel the level of antibodies to SLA/LP (anti-SLA/LP) in sera, because the titer in serum #3 was higher than that in serum #4. Thus, we found that AIH inhibited Sec synthesis but we could not conclude that this inhibition depended on an autoantibody against SLA/LP. To clarify the function of SLA/LP, we prepared an antibody against an SLA/LP peptide. Inhibition of Sec synthesis by this anti-peptide antibody was incomplete and sometimes did not occur. Absorption of autoantibodies in the AIH serum with the SLA/LP protein did not have any effects on Sec synthesis by Sec synthase (SecS) which converts Seryl (Ser)-tRNASec to Sec-tRNASec. We then used SLA/LP instead of active bovine SecS but we could not detect SecS activity in SLA/LP itself. Next, we studied the distribution of SLA/LP in HEK293 cells transfected with an SLA/LP expression vector. Immunofluorescence microscopy detected SLA/LP expression in the cytoplasm, but not the nucleus of HEK293 cells. Higher levels of SLA/LP in the cytosol did not correlate with higher Sec production, which remained identical to mock-transfected cells. Interestingly, we found that SLA/LP formed a complex in the cytosol of HEK293 cells. This complex was purified and the components analyzed using Nano-LC and MS/MS, which we identified as HSP70 and tubulin. Altogether, our data suggest that SLA/LP may play an indirect role to produce Sec in the selenoprotein synthesis machinery.

ShengMai San (SMS) comprising three herbal components (Panax Ginseng, Ophiogon japonicus and Schisandra chinensis) is one of the most popular traditional Chinese medicine prescriptions, having been used for more than 3000 years in China to treat symptoms related to Qi depletion and cardiac disorders. In the present study, antioxidant synergism among the component herbs of SMS was precisely examined in vitro using extracts prepared from respective component herbs and their possible combinations under high hydrostatic pressure (600 Mpa). Expression of antioxidant enzymes in brain was also studied in rats administered those extracts by western blotting method. It was revealed that complete SMS formula had the highest potential both in scavenging 1,1-diphenyl-2-picylhydrazyl (DPPH) and hydroxyl radicals in vitro, and also in the expression of heme oxygenase-1 (HO-1), superoxide dismutase (SOD) and cytosolic glutathione peroxidase (GPx) proteins in brain of rats administered the extract for five days. Certain synergistic interactions among the component herbs were observed in the antioxidant activity in vitro and also in the antioxidant enzyme expression in vivo. Correlation analysis between in vivo and in vitro data revealed that expression of HO-1 and SOD were highly related to in vitro DPPH and superoxide radical scavenging activities with significant correlation coefficients of 0.899 and 0.758, respectively. Expression of GPx showed weak correlation with all the in vitro antioxidant indicators. It was concluded that the combination of three herbs are essential to confer the maximal antioxidant potential to the SMS formula, and that SMS exerts potential antioxidant activity in vivo through its inherent radical scavenging activity and also the modulation of expression level of antioxidant enzymes.

Atmospheric particles at a roadside site in the downtown area of Kumamoto, Japan, were collected in the spring of 2006. A cascade impactor was used to fractionate the particles into nine size ranges: > 11.0, 11.0-7.0, 7.0-4.7, 4.7-3.3, 3.3-2.1, 2.1-1.1, 1.1-0.65, 0.65-0.43, and < 0.43 μm. Ten polycyclic aromatic hydrocarbons (PAHs) with 4, 5, and 6 rings [fluoranthene (Flu), pyrene (Pyr), benz(a)anthracene (BaA), chrysene (Chry), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), benzo(ghi)perylene (BghiP), dibenz(a,h)anthracene (DahA), indeno(1,2,3-cd)pyrene (IcdP)] in the size ranges were expressed and investigated in terms of their total and individual concentrations and the equivalents of benzo(a)pyrene (BaPE) upon their relative carcinogenic potential. The correspondence of PAHs to particle size, PAHs tending to accumulate in small size ranges, was confirmed. The mass of PAHs in the size range < 2.1 μm averaged 4-fold more than that in the range > 2.1 μm. The PAHs with 5 and 6 rings predominated in the total mass of the 10 PAHs, exhibiting approximately monomodal distributions with the mode around or smaller than 0.5 μm. The PAHs with 4 rings composed 28% of the total PAHs in mass and showed bimodal distributions with one mode in the range of < 0.5 μm and another in the range of > 1.0 μm. The estimate of BaPE using the 5- and 6-ring PAHs indicated that PAHs in particles < 2.1 μm accounted for approximately 80% of the equivalents, suggesting that the carcinogenic potential of the PAHs was dominated by small particles. BaP together with BbF, DahA, and IcdP predominated in the equivalents, while the equivalents due to BaA and BkF were very small in all size ranges.

The purpose of this research was investigating the use of an enzymatic method for identifying various treatments, such as permanent waving, hair dyeing and bleaching used on hair from hair samples. Morphologically only negligible difference was observed between untreated hair and hair permed in vitro. However, after protease treatment, the degradation of permed hair was accelerated and a significant difference was observed between permed and untreated hair in morphology and degradation extent. The degraded fraction of untreated hair was confined to cuticle region of hair surface, whereas in hair samples permed and dyed in vitro, the microfibril protein of hair was degraded. Furthermore, there was a high correlation between the extent of degradation and the hair damage resulting from these chemical treatments. When we tested the method on human hairs from live subjects, no significant difference was observed on untreated hair in different hair sections. However, the tip section of permed and dyed hair showed much higher degradation than that of root section from morphology and degradation extent. Our findings for practical uses revealed that the enzymatic method can be applied to identify the chemical treatment used on hair.

The aim of this paper is to compare the geographical routes and speeds of influenza propagation in and around Tokyo, including Kanagawa, Saitama, Tochigi and Fukushima prefectures, in the seasons of 2003/2004, 2004/2005 and 2005/2006. Each season ranges from November 1st to October 31st. The propagation pattern is estimated from the daily variations in the sales amount of anti-influenza drug at about 30 pharmacies in the area. The time lags (day) of influenza infection periods between distant pharmacies are calculated by the cross-correlation functions of the drug sales at the pharmacies. We conclude that the influenza infection spread from the urban area of Tokyo to its suburbs (Saitama and Kanagawa) along the railway systems in all the seasons examined. However, no definite infection routes can be found in Tochigi and Fukushima. The infection periods of adults are contrasted with those of children in individual observation sites.

The present investigation was undertaken to compare the structural and electronic properties of genipin with those of tetrahydrobiopterin (H4B), an essential cofactor in neuronal nitric oxide synthase (nNOS), and 4-aminotetrahydrobiopterin (4-amino-H4B), an inhibitor of nNOS, using computer-assisted molecular modeling techniques. Molecular modeling, superimposing, and docking simulation, in addition to LUMO-energy calculation, revealed that genipin has structural and electronic properties that markedly resemble those of H4B.

The complete microbial degradation of dimethyl phthalate ester (DMPE) is described. DMPE is thought to be an endocrine-disrupting chemical. A pure culture (strain No. A-9) from soil sample capable of utilizing DMPE as the sole source of carbon and energy was identified as Flavobacterium sp. Degradation patterns of DMPE were observed on the high-performance liquid chromatogram (HPLC) of the culture filtrates of this strain, and growth of bacteria was measured as protein by the Kennedy and Fewson method. The growth yield of this strain was about 5.9 g of protein per mole of carbon source of DMPE, and was similar to that in the case of glucose as a carbon source. Complete degradation of DMPE had been achieved (1000 mg/l) in less than 2 days using Flavobacterium sp. strain No. A-9. The transient intermediates of DMPE were not detectable on the HPLC of the culture filtrates of this strain. This strain also degraded phthalic acid (PA) but could not degrade phthalic anhydride.

To clarify the degradation pathway of acrinol by light, isolation and identification of acrinol degradation products (ANDP) were attempted. ANDP-2, one of the ANDPs, was isolated by extraction with methanol from cloths dampened with acrinol solution, and purified by column chromatography on Diaion HP-10, Sephadex LH-20 and G-25. The structural elucidation of ANDP-2 was examined by infrared, 1H-NMR, 13C-NMR and Fast atom bombardment mass (FAB-MS) spectra studies. From the spectroscopic data, the structure of ANDP-2 was determined to be 4-amino-6-ethoxyl-2,3-dicarboxyquinoline, that was found to be a novel degradation product of acrinol by light. The solution of ANDP-2 has fluorescence with a blue color, though that of acrinol has fluorescence with green. ANDP-2 did not show growth inhibition at a concentration of 100 μg/ml against Gram-positive, -negative bacteria, yeast and fungi.

Five strains of multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) were isolated from inpatients at a local hospital in China. The most frequent resistance was to cefoperazone, ciprofloxacin, ceftriaxone, cefotaxime, gentamicin, piperacillin, trimethoprim sulfamethoxazole, and tobramycin. These strains were found to contain the class 1 integron, in which the 2360-bp gene cassettes were flanked by 5'- and 3'-conserved segments. Sequence analysis revealed that the gene cassettes contained aacA4 and cmlA1 genes; however, the latter gene had a nonsense mutation resulting in the production of a truncated protein. To the best of our knowledge, this is the first report of a nonsense mutation in the cmlA1 gene. Moreover, the P. aeruginosa strains showed identical profiles in pulsed-field gel electrophoresis, suggesting that they were derived from the same clone. These results emphasize the importance of controlling the spread of multidrug-resistant pathogens in hospitals.

The bacteria contained in processed fresh edible sea urchin (uni in Japanese) were identified with high accuracy using 16S ribosornal DNA (rDNA) sequence analysis. The 586 isolates that were recovered from ten domestic products comprised 13 genera and 18 species. The food poisoning-related bacteria Bacillus cereus (B. cereus), Bacillus weihenstephanensis (B. weihenstephanensis), and Staphylococcus aureus (S. aureus) were detected in 4/10 samples, while the causative agents of bacterial opportunistic infections, Staphylococcus equorum (S. equorum), Stenotrophomonas maltophilia (S. maltophilia), Burkholderia cepacia (B. cepacia), and Serratia proteamaculans (S. proteamaculans), were detected in all 10 samples. It is not known whether these pathogens were components of the sea urchin microflora or were the result of contamination from the environment or human contact during the manufacturing process. Nevertheless, strict quality control standards are needed for the processing of fresh edible sea urchin. This is the first comprehensive analysis of the bacterial microflora of processed fresh edible sea urchin.

Protection against in vivo infection with Salmonella and enhancement of in vitro superoxide production by peripheral blood neutrophils are two reported effects of treatment with aqueous extracts of the fungus Rhizopus oryzae U-1 (Aq-ROU). Here, we report that Aq-ROU also has antiproliferative activity and can induce apoptosis in a human promyelocytic leukemia cell line, HL-60. During Aq-ROU-induced apoptosis, HL-60 cells undergo genomic DNA fragmentation characteristic of apoptosis after just 6 hr of treatment. Using phosphatidylserine (PS) as an indicator of apoptosis, we also found that the proportion of apoptotic cells increased significantly after 9 hr of treatment. Indeed, induction of apoptosis by Aq-ROU reached 43.3% after 24 hr of treatment, which is comparable to the effects of a known apoptosis-inducer, actinomycin D. Moreover, the activities of caspase-3, -8, and -9 increased in parallel with Aq-ROU treatment, with a peak of activity 9 hr after the initial treatment. Taken together, the results suggest that R. oryzae contains one or more water-soluble factor that can reliably and efficiently induce apoptosis in human cells via activation of caspase-3.

The effects of the dietary mannooligosaccharides (MOS) from coffee mannan on the anti-allergic functions were examined using C3H/HeN mice. The mice were given water and a dietary treatment containing 2.5% MOS ad libitum. The mice were sensitized subcutaneously with ovalbumin on the 7th, 21st, and 35th days and then sensitized intraperitoneally on the 49th day of the study. Serum samples, Peyer's patches and peritoneal exudate fluids were collected on the 50th day. In the non-ovalbumin sensitized groups, phosphate-buffered saline (PBS) was injected instead of ovalbumin. The number of peritoneal acidophils in the MOS diet-fed mice, was significantly lower than that in the control diet-fed ones. MOS treatment significantly reduced interleukin-10 production and tended to suppress ovalbumin-specific IgE in serum. However, it did not change interleukin-2 and interferon-γ production. These results suggest that dietary MOS may have an anti-allergic effect, caused by activation of cellular immunity.

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